Imaging features of localized IDH wild-type histologically diffuse astrocytomas: a single-institution case series

Isocitrate dehydrogenase wild-type (IDHwt) diffuse astrocytomas feature highly infiltrative patterns, such as a gliomatosis cerebri growth pattern with widespread involvement. Among these tumors, localized IDHwt histologically diffuse astrocytomas are rarer than the infiltrative type. The aim of this study was to assess and describe the clinical, radiographic, histopathological, and molecular characteristics of this rare type of IDHwt histologically diffuse astrocytomas and thereby provide more information on how its features affect clinical prognoses and outcomes. We retrospectively analyzed the records of five patients with localized IDHwt histologically diffuse astrocytomas between July 2017 and January 2020. All patients were female, and their mean age at the time of the initial treatment was 55.0 years. All patients had focal disease that did not include gliomatosis cerebri or multifocal disease. All patients received a histopathological diagnosis of diffuse astrocytomas at the time of the initial treatment. For recurrent tumors, second surgeries were performed at a mean of 12.4 months after the initial surgery. A histopathological diagnosis of glioblastoma was made in four patients and one of gliosarcoma in one patient. The initial status of IDH1, IDH2, H3F3A, HIST1H3B, and BRAF was “wild-type” in all patients. TERT promoter mutations (C250T or C228T) were detected in four patients. No tumors harbored a 1p/19q codeletion, EGFR amplification, or chromosome 7 gain/10 loss (+ 7/ − 10). We assessed clinical cases of localized IDHwt histologically diffuse astrocytomas that resulted in malignant recurrence and a poor clinical prognosis similar to that of glioblastomas. Our case series suggests that even in patients with histologically diffuse astrocytomas and those who present with radiographic imaging findings suggestive of a localized tumor mass, physicians should consider the possibility of IDHwt histologically diffuse astrocytomas.

7 gain and whole chromosome 10 loss (+ 7/ − 10), and/or harboring telomerase reverse transcriptase (TERT) promoter mutations should be considered true GBMs, given their poor survival prospects 9 . These LrGGs with molecular features of GBMs (i.e., features of molecular GBMs) have almost the same radiographic and histological findings as IDHmut LrGGs. The recent (2021) WHO classification of central nervous system (CNS) tumors applies molecular criteria that allow for a diagnosis of GBM, CNS grade 4, of IDHwt astrocytic gliomas, even in the absence of high-grade histopathologic features, when at least one of the following molecular features is present: concurrent EGFR amplification, chromosome 7 gain/10 loss (+ 7/ − 10), or a TERT promoter mutation 10 . Regarding the overall survival (OS) of IDHwt diffuse astrocytomas, one group has reported that median OS of the patients with IDHwt diffuse astrocytomas meeting the definition for molecular GBM was 42 months, which had no significant difference compared with patients with IDHwt diffuse astrocytomas not meeting this definition (median OS: 57 months) 11 . In contrast, another group has revealed that the median OS for IDHwt diffuse astrocytomas, grade 4 (based on the 2021 WHO classification of CNS tumors) and IDHwt glioblastomas was similar (23.8 months vs 19.2 months) 12 . Therefore, the exact prognosis of IDHwt diffuse astrocytomas remains unknown.
Some studies have reported that localized types of molecular GBMs are rarer than the infiltrative type [13][14][15] . Moreover, both IDHwt grade II astrocytomas and IDHwt grade II astrocytomas meeting the definition of molecular GBM (according to the 2016 WHO classification of CNS tumors) were reported to possess highly infiltrative patterns, such as gliomatosis cerebri growth patterns with widespread involvement. 11 Although localized tumors are typical in LrGGs, it is not clear whether this radiographic feature affects the clinical prognosis of IDHwt LrGGs.
The authors analyzed five cases of localized IDHwt histologically diffuse astrocytomas and found that they all resulted in malignant recurrence and a poor clinical prognosis similar to that of GBMs. Although a number of studies have investigated IDHwt LrGGs [11][12][13]16,17 , very few studies have reported detailed information on localized IDHwt LrGGs. In the present study, we therefore describe the clinical, radiographic, histopathological, and molecular characteristics of localized IDHwt histologically diffuse astrocytomas.

Results
The five patients' clinical characteristics are summarized in Table 1. All patients were female, and their mean age at the time of the initial treatment was 55.0 years. The tumors were located in the insula in three patients (60.0%), in the precentral gyrus in one patient (20.0%), and in the frontal operculum in one patient (20.0%). The tumors were located in the left hemisphere in four cases (80.0%) and in the right hemisphere in one case (20.0%) (Fig. 1). All patients had focal disease, not including gliomatosis cerebri or multifocal disease. All initial tumors showed no contrast enhancement on Magnetic Resonance Imaging (MRI). Gross total resection (GTR) was achieved in three patients and partial resection (PR) in two patients. The pathological diagnosis in all cases  Thus, no adjuvant chemoradiotherapy was performed. Recurrent tumors developed in or near the surgical defects in all patients, and all tumors showed contrast enhancement on MRI. The second surgery was performed in all patients, at a mean of 12.4 months after the initial surgery (progression-free survival; PFS). The median overall survival (OS) of five patients was 28.7 months (95% CI, 19.0 to 38.4). A histopathological diagnosis of GBM was made in four patients and one of gliosarcoma in one patient ( Table 1). As treatment at recurrence, all patients underwent adjuvant temozolomide and concurrent local radiotherapy or gamma knife radiosurgery. Two patients underwent a third surgery for recurrent tumors. Bevacizumab was additionally prescribed to three patients after the second or third surgery. Table 2 shows the molecular characteristics of the initial and recurrent tumors. The status of IDH1, IDH2, H3F3A, HIST1H3B, and BRAF was "wild-type" in all patients. TERT promotor mutations (C250T or C228T) were detected in four patients. No tumors harbored 1p/19q codeletion. EGFR amplification, or chromosome 7 gain (+ 7) was detected in one patient at only recurrence. CDKN2A/B homozygous deletion was detected in two patient and PTEN losses were detected in one patients. According to these molecular details, four patients showed the high-grade molecular features of GBM as defined by the cIMPACT-NOW update 3 criteria 9 .

Case presentation (Case 3).
A 58-year-old woman presented with paresthesia of the right side of her body and visited a nearby hospital. She underwent brain MRI that revealed a T2-weighted high-intensity lesion, mainly in the left frontal lobe (Fig. 3A). The patient was referred to our hospital for further investigation and treatment. On her first visit to the outpatient clinic, she had no abnormal neurological findings, including palsy or aphasia. Brain MRI detected a low-intensity lesion on T1-weighted images with no gadolinium enhancement and a high-intensity lesion with relatively well-marked boundaries on T2-weighted images in the left inferior frontal gyrus, with a maximum diameter of approximately 30 mm (Fig. 3A,B). The patient underwent awake craniotomy assisted by both cortical and subcortical functional mapping. Functional mapping was performed with a double task combining picture-naming and right-arm movements. GTR was achieved (Fig. 3C). Postop-  www.nature.com/scientificreports/ eratively, the patient showed no remarkable neurological deterioration in language abilities and movement of the upper and lower extremities. The histopathological diagnosis was diffuse astrocytoma (Fig. 4A). Immunohistochemistry staining for IDH1 R132H was negative (Fig. 4B). Molecular analysis revealed the TERT promoter mutation C228T and CDKN2A/B homozygous deletion. No postoperative adjuvant therapy was administered. Approximately 6 months later, follow-up contrast MRI showed a well-enhancing mass lesion in the left frontal surgical cavity (Fig. 3D). The patient underwent awake surgery for the recurrent tumor, and GTR was achieved (Fig. 3E). Histological examination of the tumor yielded a diagnosis of GBM, and Ki-67 expression was observed in 5-7% of the tumor cells ( Fig. 4C). At the recurrent stage, immunohistochemical staining of IDH1 R132H was also negative (Fig. 4D).
Following the second surgery, the patient was treated with 60 Gy in 30 fractions of radiotherapy in combination with concurrent oral TMZ chemotherapy (standard Stupp protocol). Furthermore, the patient received a Table 2. Molecular alterations observed in five patients with initial and recurrent localized IDH wild-type histologically diffuse astrocytomas. IDH isocitrate dehydrogenase, TERT telomerase reverse transcriptase, BRAF v-raf murine sarcoma viral oncogene homolog B1, EGFR epidermal growth factor receptor, CDKN2A/B cyclin-dependent kinase inhibitor 2A/B, PTEN phosphatase and tensin homolog deleted on chromosome 10, TP53 TUMOR PROTEIN p53, CDK4 cyclin-dependent kinase 4, MDM2 mouse double minute 2, + 7; whole chromosome 7 gain, − 10; whole chromosome 10 loss, hemi; hemizygous deletion.  www.nature.com/scientificreports/ maintenance regimen of TMZ 200 mg/m 2 on days 1-5 every 4 weeks. Four months after the second surgery, follow-up MRI revealed a recurrent lesion on the posterior side of the surgical defect, and bevacizumab was additionally prescribed (Fig. 3F). However, the recurrent lesion showed progression, and a third craniotomy for tumor removal was performed 9.5 months after the second surgery; GTR was achieved (Fig. 3G). The histopathological diagnosis changed from diffuse astrocytoma to GBM. Stereotactic radiotherapy (40 Gy) was performed postoperatively, and chemotherapy, including temozolomide and bevacizumab, was continued. Despite multidisciplinary treatment, tumor progression was not controlled (Fig. 3H). The patient decided to leave the hospital to receive palliative care at home, and she died approximately 23 months after being diagnosed with diffuse astrocytoma.

Discussion
In the current study, we present a single-institution retrospective series of five patients who were diagnosed with localized IDHwt histologically diffuse astrocytomas. We provide their clinical information, radiographic characteristics, genetic alterations, and treatments such as surgery and adjuvant therapy. The radiographical and pathological features of the initial tumors in our case series were almost identical to those of diffuse astrocytomas, IDHmut (CNS WHO grade 2). All tumors relapsed at a mean of 12.4 months after initial surgery, resulting in a dismal prognosis despite multidisciplinary treatment.
Among the five cases presented here, there were four patients with molecular features of GBM, which represents a new entity proposed by the cIMPACT-NOW committee to classify a subset of IDHwt diffuse or anaplastic astrocytomas into a "GBM, IDHwt, CNS WHO grade 4" category 12,18 . These four cases harbored TERT promoter mutations. TERT promoter mutations are known to be molecular markers of an aggressive phenotype, with which even LGGs behave like GBMs, and the pathological diagnosis of such cases is GBM-IDHwt, according to the 2021 WHO classification of tumors of the CNS 10 . Although these four tumors exhibited radiological and histopathological features similar to IDHmut, CNS WHO grade 2 tumors, they presented with an aggressive clinical course leading to poor survival, more similar to patients with both histological and molecular GBM, IDHwt, www.nature.com/scientificreports/ CNS WHO grade 4. Moreover, the exact prognosis of IDHwt LrGGs that do not meet the definition of molecular GBM is unknown. One retrospective study reported that the OS associated with IDHwt LrGGs not meeting the definition of molecular GBM is significantly longer than that associated with molecular GBMs 12 , while another study reported that there was no significant difference between the prognoses for these two groups 11 . Our patient (Case 4) did not meet the definition of molecular GBM and relapsed 10 months after partial resection during the initial surgery. The second pathological diagnosis was one of GBM. The clinical course of this patient did not seem to significantly differ from that of other cases of molecular GBM (Cases 1-3,5). The initial tumor of Case 4 may have been a lower-grade astrocytoma, not a GBM, at initial diagnosis, and it may have subsequently harbored CDKN2A/B homozygous deletions at recurrence 10 months after the first surgery. Based on clinical data, radiographic imaging, and histopathology only, tumors with molecular features of GBM are difficult to distinguish from IDHmut LGGs. Although IDHwt histologically diffuse astrocytomas are uncommon, some studies have revealed that they are especially rare in women, and that they are associated with older age at initial diagnosis, fronto-temporo-insular location, and highly invasive behavior 3,5,7 with frequent infiltration of the adjacent cortex and deep white matter 19 . Therefore, IDHwt histologically diffuse astrocytomas possess features of highly infiltrative patterns, such as gliomatosis cerebri growth patterns with widespread involvement 2 .
Data on tumor growth patterns in patients with IDHwt grade II or III LrGGs from studies published in the literature between 2018 and 2021 were extracted and compiled as shown in Table 3 [11][12][13][14]17,[20][21][22][23] . In the cIMPACT-NOW update 3 report 12 , Tesileanu et al. reported that a gliomatosis cerebri growth pattern of LrGGs was present in 35.8% of IDHwt astrocytomas, meeting the definition of molecular GBM. Furthermore, this pattern had a high frequency of 52.4% in IDHwt astrocytomas, meeting the definition of molecular GBM with only TERT promoter mutations. In their study of 47 patients with IDHwt diffuse astrocytomas, Berzero et al. found that infiltrative gliomatosis cerebri growth patterns were detected in 72% of IDHwt diffuse astrocytomas. In particular, these patterns were found in 73% of IDHwt diffuse astrocytomas, meeting the definition of molecular GBM 11 . In our series, five cases (45.4%) presented with a localized tumor mass without a multifocal or gliomatosis-cerebri pattern. Conversely, six patients (54.5%) presented with tumors exhibiting gliomatosis-cerebri growth patterns. From the point of view of the tumor border line, tumors with sharp or well-defined borders account for 0-33% and tumors with indistinct or ill-defined borders account for 38-100% of all tumors. In a systematic review and meta-analysis, Lent et al. reported that IDHwt astrocytomas showed significantly fewer sharp borders than Table 3. Literatures about IDH wild-type lower grade gliomas. IDH isocitrate dehydrogenase, wt wild-type, EOR extent of resection, OS overall survival, R retrospective study, pTERT telomerase reverse transcriptase promoter, EGFR epidermal growth factor receptor, NOS not otherwise specified, N/A not available, CT chemotherapy, RT radiotherapy, CRT chemoradiotherapy, OD oligodendroglioma, OA oligoastrocytoma, GTR gross total resection, PR partial resection. www.nature.com/scientificreports/ IDHmut astrocytomas (29% and 50%, respectively) 15 . Thus, localized IDHwt lower grade astrocytomas, such as those described here, are considered rare. Therefore, even in a patient presenting with radiographic imaging findings suggestive of a localized tumor mass, physicians should remain aware of the possibility of lesions representing IDHwt histologically diffuse astrocytomas. Surgery of localized tumors tends to achieve greater extent of resection (EOR) than resection for infiltrative tumors, and it has been reported that a greater EOR is associated with better outcomes for IDHwt LrGGs 24,25 . Moreover, some studies have reported that IDHwt grade II gliomas have a better prognosis than IDHwt grade III gliomas 5,11,26 . While this suggests that localized IDHwt grade II gliomas also have a better prognosis, contrary to our expectations, our cases showed an extremely aggressive clinical course. In Cases 1 and 3, the tumor relapsed only 7.5 and 5.8 months after initial surgery, even after GTR had been achieved. All recurrent tumors in our cases developed in or near the surgical defects and retained their localized radiological features. In four cases (Cases 1-3, 5), recurrent tumors showed resistance to multidisciplinary treatment and resulted in progression. Only one case (Case 4) was undergoing a maintenance regimen of TMZ without progression. Our findings suggest that IDHwt histologically diffuse astrocytomas need to be followed up very cautiously, even after gross total removal, and that strong postoperative adjuvant therapy such as the Stupp protocol for GBM should be considered.
Taken together, there is a strong need for routine analysis for molecular features of GBM, even for seemingly low-grade components. Several studies have reported on non-invasive imaging modalities to predict IDH or TERT promoter mutation status to guide treatment strategies for gliomas from the preoperative stage of the initial clinical diagnosis. 27,28 To predict the IDH status of gliomas from preoperative MRI, Choi et al. 28 developed a model based on deep learning and radiomics using a fully automated hybrid approach. The authors demonstrated that their approach allows for the accurate prediction of IDH status of gliomas. Lu et al. 27 presented a radiomics feature-based nomogram for predicting TERT promoter mutation status from preoperative MRI in patients with lower-grade glioma. The radiomics signature yielded good performance for predicting TERT promoter mutation status, with a high area under the curve (AUC) of 0.900 and 0.873 in the training and validation datasets, respectively. These modalities provide promising methods for preoperatively predicting molecular features of gliomas and have the potential to guide treatment strategies for patients with gliomas.
Although we provide novel information on localized IDHwt histologically diffuse astrocytomas, our results are somewhat limited, compared to those of prospective studies, as retrospective studies may be influenced by unrecognized biases. Moreover, the present study was based on a small number of tumor cases; therefore, a larger cohort study is needed to assess the clinical, radiographic, histopathological, and molecular characteristics of localized IDHwt histologically diffuse astrocytomas. Thus, further accumulation of evidence for IDHwt histologically diffuse astrocytomas will help improve the treatment of this disease and hopefully enable us to develop it into a novel therapy in the future.
Another limitation of this study is that the initial radiological diagnosis of diffuse astrocytoma could have been changed to high-grade glioma, including molecular features of GBMs, using advanced MRI techniques such as diffusion, perfusion, spectroscopy, tractography, and functional MRI 29 . Moreover, another limitation of our study was that we were unable to perform a second MRI approximately 6-12 weeks after the first diagnosis. This would have allowed for the detection of a high-grade glioma the radiological tumor growth rate, which is a strong indicator of tumor aggressiveness 30 . Repeat MRI could detect the tumor's features, even if the initial MRI presentation was in favor of low-grade gliomas. Thus, "multimodality" imaging should have been performed after the initial diagnosis to allow for a revision of the initial diagnosis of low-grade gliomas to high-grade gliomas.

Conclusions
Although IDHwt histologically diffuse astrocytomas possess highly infiltrative patterns, such as gliomatosis cerebri growth patterns with widespread involvement, we observed clinical cases of localized IDHwt histologically diffuse astrocytomas, which resulted in malignant recurrence and a poor clinical prognosis similar to that of GBMs. Even in patients with histologically diffuse astrocytomas and those who present with radiographic imaging findings suggestive of a localized tumor mass, physicians should consider the possibility of IDHwt histologically diffuse astrocytomas.

Materials and methods
Patient data. We have totally experienced eight cases of IDHwt diffuse astrocytoma and thirteen cases of IDHmut diffuse astrocytoma between July 2017 and January 2020 at Nagoya University Hospital (Nagoya, Japan). Of these, the data of five patients who were diagnosed and treated for localized IDHwt diffuse astrocytoma were retrospectively retrieved from patient records. A localized tumor mass was defined as one with a welldefined tumor border, where the border between the tumor and normal brain can be delineated on T2-weighted MRI. Moreover, "localized tumors" do not present with gliomatosis cerebri growth patterns with widespread involvement or multifocal disease. Surgery was proposed with the aim of maximal tumor resection and preservation of neurological functions. In all five patients, we performed awake brain mapping with direct electrical stimulation using an asleep-awake-asleep protocol, as previously described [31][32][33][34][35][36] . Patient data on clinical information and outcomes, including age, sex, histopathological findings, prescribed treatment, EOR, radiographic findings before and after treatment, molecular features of the tumors, PFS and OS were collected and analyzed. Volumetric EOR was categorized as follows: GTR, EOR = 100%; subtotal resection (STR), EOR ≥ 90% to < 100%; and PR, EOR < 90%. Histopathological diagnoses had been established independently, by histological confirmation according to the 2016 WHO criteria 2 , by two expert neuropathologists (Y.S, M. N).
Statistical analyses. All statistical analyses were conducted using SPSS (version 27.0; IBM Corporation, Armonk, NY, USA) for Windows (Microsoft Corporation, Redmond, WA, USA). PFS was determined, from the day of the first surgery until the occurrence of true tumor progression before the second surgery. OS was calculated from the date of the first visit to the date of death or last follow-up.
Ethics approval and consent to participate. The Ethics Committee of Nagoya University Hospital approved the data evaluation (Approval Number: 2021-0410). All procedures performed in studies involving human participants in accordance with all provisions of the Declaration of Helsinki. Patient informed consent was waived due to the retrospective nature of the study approved by the Ethics Committee of Nagoya University Hospital.

Data availability
The data in the current study are available from the corresponding author upon reasonable request.

Code availability
Not applicable.